Atrial fibrillation (AF) increases risk of heart failure, stroke and death, and its incidence increases with age. Genome wide association studies (GWAS) have identified 14 genetic loci associated with AF, but the causal genetic variant(s) and the mechanisms by which these variants increase AF risk remain unknown. Our overall goal is to identify functional variants in AF-associated loci that alter gene function or expression, and t elucidate the impact on associated biological pathways, thus discovering potential novel targets for AF therapy or prevention. AF is characterized by triggers (ectopy) that initiate AF and atrial substrates that promote AF propagation. Triggers usually originate in left atrial (LA) pulmonary vein (PV) ostia, while ion channel and structural remodeling with altered protein and cellular composition can occur throughout the atria. Many GWAS-identified AF loci are in non-coding regions near genes that may contribute to embryologic PV formation or electrical triggering, as well as to cellular changes that might promote substrates that propagate AF. Our genetic-transcriptomic studies via next generation RNA sequencing (RNAseq) in human adult left atrial appendage tissues have identified single nucleotide polymorphism (SNP) effects on gene expression (expression quantitative trait loci, eQTLs) for 6 of 14 AF GWAS loci that can now be targeted to identify the causal genetic variant. However, we did not find an eQTL for the top GWAS locus near PITX2; this gene may be most active during embryologic development in the PV-LA junction, at an earlier time and in a different atrial region than we studied in adult LAA tissues. Here we seek to advance our discoveries via 2 aims focused on: 1) Functional studies for 3 AF GWAS loci for which we identified strong eQTLs; and 2) Genetic-transcriptomic studies in the PV-LA junction region, the main site of AF triggers, and in induced cardiomyocytes derived from stem cells (iCMs) to discover eQTLs for additional AF GWAS loci, including the PITX2 locus, which can lead to targets for future functional studies. We will perform functional studies of the top 3 eQTLs in AF GWAS loci in iCMs and determine the cellular effects of under- or over-expression of these eQTL genes. Pathophysiologic mechanisms by which the 3 top eQTLs predispose to AF will be evaluated in iCMs through functional genomic, cell biological, electrophysiological and metabolic studies that utilize reporter gene transfection, siRNA knockdown, transfection-mediated over expression, and genome editing with the CRISPR-Cas9 system. We will evaluate tissues from the PV-LA junction and iCMs during differentiation and maturation for expression of AF-associated genes with the goal of identifying additional eQTLs that may underlie AF susceptibility. We will also identify genes and pathways whose expression and activity are regulated by genetic variants. By achieving the aims of this proposal we will pinpoint the specific mechanisms and targets of loci identified by genomic analyses of AF, bringing us closer to clinical application of genomic findings.